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Quaternary Ammoniums and Other Preservatives’Contribution in Oxidative Stress and Apoptosison Chang Conjunctival Cells

Caroline Debbasch,1,2 Franc ¸oise Brignole,3 Pierre-Jean Pisella,1,2 Jean-Michel Warnet,1

Patrice Rat,1 and Christophe Baudouin2

P URPOSE. To investigate some of the toxicity mechanisms of 10preservatives currently used in ophthalmic solutions in vitro.

METHODS. A continuous human conjunctival cell line wastreated with different concentrations of various preservativesfor 15 minutes and for 15 minutes followed by 24 hours of cellrecovery: three benzalkonium chlorides (BACs) with differenthydrocarbon chain length, benzododecinium bromide (BOB),cetrimide (Cet), phenylmercuric nitrate (PM), thimerosal (thi),methyl parahydroxybenzoate (MPHB), chlorobutanol (clb),and EDTA. An inhibition study was then conducted using a1-hour vitamin E pretreatment followed by a 15-minute BACtreatment. Membrane integrity was assessed using a neutral redtest and chromatin condensation with a Hoechst 33342 test.Reactive oxygen species were measured using dichlorofluores-

cein diacetate test for H2O2 production and hydroethidine testfor O2

. production. These tests were performed using micro-plate cold light cytofluorometry. Cell size and DNA content

were also analyzed using flow cytometry. Confocal microscopy was used to explore morphologic changes.

R ESULTS. A significant decrease of membrane integrity with chromatin condensation was observed with all the quaternary ammoniums tested at concentrations of 0.005% and higher.The effect was amplified after 24 hours of cell recovery. Theother preservatives tested did not decrease membrane integ-rity. H2O2 production was observed with all the preservatives,

whereas O2. production was significantly higher with the qua-

ternary ammoniums at 0.005% and 0.01%, compared with theother preservatives. Flow cytometry results confirmed the cy-totoxicity observed with cold light cytofluorometry.

CONCLUSIONS. The quaternary ammoniums tested (BAC, BOB,and Cet) were the most cytotoxic preservatives in the currentmodel. An apoptotic mechanism appeared to be present at low concentrations of quaternary ammoniums, whereas a necroticprocess appeared at higher concentrations. Superoxide anionsmay play an important role in tissue damage induced by pre-servatives in ocular surface disorders. ( Invest Ophthalmol VisSci. 2001;42:642–652)

Preservatives are used in most ophthalmic preparations,including eye drops and contact lens solutions. Although topically administered medications are increasingly used with

apparent safety and good tolerance, there is growing evidencethat long-term use of topical drugs can induce changes in theocular surface and may often produce damage to conjunctivaland corneal epithelial cells. There have been several reports of the toxic effects of prolonged topical treatments, partly due tothe preservatives associated in the formulation of such treat-ments.1–3 In the eye, preservative turnover is very slow, andquaternary ammonium molecules can be retained in ocular tissues up to 7 days.4 The lipophilic nature of some preserva-tives causes them to bind to the ocular tissues immediately after topical application. Previous studies by Burstein3 haveshown that topically applied benzalkonium chloride (BAC), themost commonly used preservative in ophthalmic solutions, cancause morphologic disruption of the corneal epithelium at

high concentrations.3

In addition, there is evidence that clini-cal concentrations of BAC may change the ionic resistance of the cornea by intercalating into cellular membranes, which results in increased permeability.5 Three types of mechanismshave been described: detergent effects causing loss of tear filmstability, toxic effects to the corneal and conjunctival epithelia,and immunoallergic reactions.2,6,7 Furthermore, repeateddoses of preserved eye drops can lead to a cumulative effect,because the preservatives are in prolonged contact with theepithelium. Several studies have confirmed the participation of preservatives in induction of ocular surface inflammation,8,9

allergy,6 fibrosis,10 and dry eye syndrome.11,12 Preservativesare also suspected of strongly increasing the risk of failure of trabeculectomy in glaucoma.13–16

In vitro models have been developed to predict the cyto-

toxic potential of preservatives. These models were essentially based on corneal epithelial cells17,18 or on other epithelialsystems with characteristics similar to those of the superficiallayer of the corneal epithelium (Madin–Darby canine kidney cells).19 The human continuous conjunctival cell line has alsobeen useful for ocular toxicological studies.20–22 We recently showed that BAC is a strong proapoptotic agent in Chang’sconjunctival cells.23

The purpose of this study was to investigate, by flow cy-tometry and microplate cold light cytofluorometry, the cyto-toxicity of 10 of the most common preservatives used inophthalmic solutions. Because stimulation of reactive oxygenspecies (ROS) constitutes one of the mechanisms of cytotox-icity, we investigated ROS production induced by preservativesin a well-adapted cellular model for in vitro cytotoxicity. More-

over, the protective effect of vitamin E against the cytotoxicity of preservatives was explored. The new technique of micro-plate cold light cytofluorometry for cytotoxicity assays hasbeen validated in Chang conjunctival cells in previous stud-ies23–25 and allows the use of numerous fluorescent probesdirectly on living cells. Their specificity, sensitivity and stan-dardization ensure that they are well-adapted to cellular heter-ogeneity and comply with the requirements of cellular phar-macotoxicology screening procedures. Thus, this study couldbe better performed physiologically on live rather than deadcells, because labile markers can be significantly affected by

From the 1Unit of Cellular Pharmacotoxicology, Centre Hospi-talier National d’Ophtalmologie des Quinze-Vingts, the Toxicology Laboratory, University of Paris-V; and the 2Ophthalmology and 3Immu-nohematology Services, Hôpital Ambroise Paré, Assistance Publique–Hôpitaux de Paris, University of Paris-V, Boulogne, France.

Submitted for publication August 17, 2000; revised October 27,2000; accepted November 2, 2000.

Commercial relationships policy: N.Corresponding author: Christophe Baudouin, Service d’Ophtal-

mologie, Hôpital Ambroise Paré, AP-HP, Université Paris-V, 9 avenueCharles de Gaulle, 92104 Boulogne cedex, [email protected]

Investigative Ophthalmology & Visual Science, March 2001, Vol. 42, No. 3

642 Copyright © Association for Research in Vision and Ophthalmology


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the use of extraction techniques. Therefore, we analyzed, inthe Chang’s human continuous conjunctival cell line, the im-mediate and delayed actions of different concentrations of preservatives on membrane integrity, cell size, DNA conden-sation, and ROS production. Variations of labile markers (ROS)

were therefore instantaneously detected, thereby providingreliable data. To our knowledge, this is the first report todescribe the relation between oxidative stress and apoptosisafter preservatives treatments and to compare in a similar

biologic way the cytotoxicity of 10 different preservatives.Results thus obtained on the human conjunctival cell line may contribute to a better understanding of preservative cytotox-icity.

M ATERIALS AND METHODS

Conjunctival Cell Line

Wong–Kilbourne-derived human conjunctival epithelial cells, an estab-

lished cell line (Wong–Kilbourne derivative of Chang conjunctiva,

clone 1-5c-4, American Type Culture Collection [ATCC] certified cell

line [CCL], 20.2), were cultured under standard conditions (humidified

atmosphere of 5% CO2 at 37°C) in Dulbecco’s minimum essential

medium (DMEM; Eurobio, Les Ulis, France) supplemented with 10%

fetal bovine serum (Dominique Dutscher, Brumath, France), 1% glu-tamine (Eurobio), 0.1% ampicillin (Panpharma, Fougères, France), and

2% kanamycin (Bristol Myers Squibb, Paris, France). Cells from pas-

sages 6 through 17 (after ATCC initial passage 65) were used in all

experiments. Normal culture development was assessed daily by

phase-contrast microscopy. Confluent cultures were removed by gen-

tle trypsin incubation, and cells were counted. They were then seeded

into 96-well culture plates for microtitration analysis (5,000 cells per

well; [Nunc, Roskild, Denmark] and plated in 15-cm2 flasks (Nunc) for

flow cytometric analyses. Cultures were kept at 37°C for 24 hours.

After subconfluence was attained (culture surface covering nearly

70%), cells were exposed to the different formulations. Because this

cell line spontaneously undergoes apoptosis at 100% confluence,25

70% of confluence was thus chosen to avoid any artifact in membrane

integrity assays.

Preservative Treatments

Five different preparations of quaternary ammonium molecules were

examined. Three formulations of BAC were tested: (1) C14 benzal-

konium chloride, alkyl dimethylbenzylammonium chloride (100%

14-carbon alkyl; BAC100 ); (2) C14/C1 2 benzalkonium chlorid e (58%

14-carbon alkyl; 32% 12-carbon alkyl; BAC58 ); and (3) C14/C1 2

benzalkonium chloride (32% 14-carbon alkyl; 58% 12-carbon alkyl;

BAC32 ). Their antise ptic action is effect ive at low concentration s

and is due to the hydrocarbon chain length. The maximum activity

is obtained with the C14 molecules and the minimum with the C8

and C18 preservatives.26 In eye drops, the precise composition of

BAC is almost never known, because a mixture of C12 and C14

chains is mostly used. We therefore decided to compare the even-

tual difference in toxic effects of various preparations of BAC that

have different antiseptic activities.Benzododecinium bromide (BOB) and cetrimide (Cet) were also

tested. The five quaternary ammoniums were each tested at concen-

trations of 0.00001%, 0.0001%, 0.001%, 0.005%, and 0.01%, the con-

centration used in most eye drops being 0.01%.

Five other preservatives were tested at five concentrations: (1)

phenylmercuric nitrate (PM) at concentrations ranging between

0.000001% and 0.001%, its usual concentration being 0.001%; (2)

thimerosal (thi) at concentrations ranging between 0.000004% and

0.004%, usually used at 0.004%; (3) methyl parahydroxybenzoate

(MPHB) at concentrations ranging between 0.00003% and 0.03%, its

usual concentration being 0.03%; (4) chlorobutanol (clb) at concen-

trations ranging between 0.00005% and 0.05%, its usual concentration

being 0.5%, because high concentrations of this drug are required to

produce antimicrobial effects, and we could not obtain this concen-

tration because other excipients are needed to make clb soluble; and

(5) EDTA at concentrations ranging between 0.00001% and 0.01%, the

most common concentration used being 0.01%.

An inhibition study was performed using a 1-hour vitamin E pre-

treatment followed by a 15-minute BAC 0.001% treatment.

All preservatives and vitamin E were provided by Transphyto,

Clermont-Ferrand, France. All dilutions were realized in culture me-

dium. The complete culture medium was used as a negative control.

Durations of cell treatments with preservatives were chosen as acompromise between in vitro and in vivo data currently available on

preservatives and in line with our previous work using the same

conjunctival cell line. In vitro, it has been demonstrated that a 100-

second application of 0.007% BAC produces lysis of 50% of conjunc-

tival cells.27 A 1-hour application of 0.0013% to 0.007% BAC solution

on epithelial corneal cells also produces a 50% decrease in membrane

integrity.28 In vivo, in corneal and conjunctival tissues, BAC has a

half-life of 20 hours for the epithelium and 11 hours for the total

conjunctiva.4

In the present study two incubation times were therefore applied

to control and treated cells: 15 minutes of treatment and 15 minutes

followed by 24 hours of cell recovery in normal culture medium, as

performed in our previous studies.23–25 The 24-hour cell recovery

period was also tested as a way of approaching the clinical conditions

in which the conjunctival tissue may recover after eye drop instillation.

Experimental Procedures

Experiments were performed using microplate cold light fluorometry,

which allows fluorometric detection (280– 870 nm) with high sensi-

tivity (picograms to femtograms per milliliter) and specificity. Fluorom-

etry was performed with a microplate cytofluorometer 29 (Fluorolite

1000; Dynex; Cergy Pontoise, France). According to the recommenda-

tions of the European Centre for the Validation of Alternative Methods

(ECVAM), three cellular markers were evaluated: cellular viability,

cellular proliferation, and cellular metabolism with ROS production.30

To complete these results, cell size and DNA content were also

analyzed by flow cytometry. All flow cytometric measurements were

performed on a commercially available flow cytometer (EPICS XL;

Beckman Coulter, Miami, FL) equipped with an argon laser emitting at

488 nm, using software provided by the manufacturer (EPICS XLsystem II; Beckman Coulter) for data analysis.

Microplate Cold Light Fluorometry. This new and originaltechnique allows direct use of numerous fluorescent probes directly

on living cells and allows analysis of 96 wells in less than 1 minute.

Furthermore, each cell sample can be considered sufficiently similar to

the other samples.29 All fluorescent probes were added to live cells, in

mostly physiological conditions, because this method allows detection

of the fluorescent signal directly in the microplate cytofluorometer.

Four different tests were used according to previously validated

methods in a Chang’s cell line20,22 and other cell systems.31,32 Briefly,

membrane integrity, closely correlated with cellular viability, was eval-

uated with neutral red (Fluka, Ronkonkoma, NY) using fluorometric

detection (excitation, 535 nm; emission, 600 nm). Neutral red was

used at 50 g/ml. In accordance with the validated protocol of Boren-

freund and Puerner,33

200 l per well of medium containing neutralred was added to living cells, and the microplates were incubated for

3 hours at 37°C in atmosphere with 5% CO2. The neutral red fluores-

cence was measured as previously described.29

H2O2 was detected with the 2,7-dichlorofluorescein diacetate

(DCFH-DA; Molecular Probes, Eugene, OR) dye added to live cells

before any treatment, as previously described.32 This probe is a non-

fluorescent cell-permanent compound currently used in flow cytom-

etry that we adapted to microplate cytometry. Once inside the cell, it

is cleaved by endogenous esterases and can no longer pass out of the

cell. The de-esterified product becomes the fluorescent compound

2,7-dichlorofluorescein on oxidation by ROS. The fluorescent signal

detected (excitation, 490 nm; emission, 535 nm) has been demon-

strated to be proportional to ROS production.31,32

IOVS, March 2001, Vol. 42, No. 3 Preservatives and Conjunctival Cells 643


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O2. was detected using hydroethidine (Molecular Probes). It was

oxidized to the fluorescent ethidium cation by O2., allowing the cation

to bind to nuclear DNA with an extensive fluorescent enhancement.34

The probe was used on cells at 5 M after 10 minutes (excitation, 485

nm; emission, 600 nm).

Hoechst 33342 (Molecular Probes) is a specific UV fluorescent

probe (excitation, 360 nm; emission, 450 nm). It specifically reacts

with the DNA, at adenine and thymine levels, by intercalation after 30

minutes.35,36 This probe was used on cells at a final concentration of 10

g/ml. One microliter of propidium iodide (Sigma, St Louis, MO) at 0.5mg/ml was added to the Hoechst 33342 solution to control necrosis of

cells. In all experiments, the background fluorescence was determined

on wells without cells but containing the dye solution and was de-

duced from all control and treated wells.

Microplate cold light cytofluorometry results were obtained in

fluorescence units and were expressed as a percentage of the control.

Wells containing cells with complete culture medium but without any

treatment were used as the control. Each drug concentration was

tested in six wells, and each experiment was performed in triplicate.

Statistical comparisons were performed using an analysis of variance

(ANOVA) test to compare the five quaternary ammoniums. The Mann–

Whitney test and the z correlation test at a 0.05 level of significance

were also performed (Statview IV for Windows; Abacus, Berkeley, CA).

Flow Cytometric Analysis. Alteration of cell size after preser-

vative treatments was confirmed with flow cytometric analysis of forward scatter on a linear mode performed 15 minutes after the

treatment. Cells were trypsinized, washed with cold phosphate-buff-

ered saline (PBS), and analyzed for size. At least 3000 cells were

analyzed per sample.

DNA Content. After 15 minutes of treatment, cells weretrypsinized, washed with cold PBS, and fixed 10 minutes with 95%

ethanol in PBS at 20°C. Samples were washed with cold PBS, stained

with propidium iodide at room temperature for 20 minutes, and

analyzed on the flow cytometer. The sub-G1 region was determined by

a gate defined in the controls in the whole-cell population, as described

previously.20,37

Immunocytology. In parallel, standard immunofluorescence was performed to assess morphologic patterns of cells. Cells were

cultured on slides and treated with preservatives for 15 minutes. They

were washed with PBS and fixed as previously described. Phalloidin(Alexa 488; 200 units/ml, Molecular Probes) was then added to explore

for F-actin. After 30 minutes of incubation, cells were washed in PBS.

Propidium iodide was added to mark cell nuclei before examination

with a confocal epifluorescence microscope (E800 PCM 2000; Nikon,

Tokyo, Japan).

R ESULTS

Cellular Viability and MembraneIntegrity Evaluation

Membrane integrity significantly decreased after 15 minutes of treatment with all the quaternary ammoniums tested (Fig. 1A).This toxicity appeared at concentrations of 0.005% and 0.01%,

and membrane integrity decreased between 20% and 36% of the control value ( P 0.001 compared with control for allpreservatives). After 24 hours of cell recovery (Fig. 1B), signif-icant cellular damage was found at 0.001% and above. Thesame decrease of membrane integrity was observed with BAC100, BAC58, and BAC32, ranging between 70% with 0.001%BAC to 30% with 0.01% BAC. With BOB, this decrease variedfrom 56% at 0.001% to 32% at 0.01%, and with Cet, it variedfrom 57% at 0.001% to 31% at 0.01%. No significant difference

was found when the five quaternary ammoniums were com-pared using ANOVA.

Clb, EDTA, organomercurials (thi and PM), and MPHB didnot decrease membrane integrity after 15 minutes or after 24hours of cell recovery for all the concentrations tested (data

not shown). In Table 1 we present the mean values obtained with all the concentrations tested for Clb , EDTA, organomer-curials, and MPHB (no significant differences between the fiveconcentrations tested) and the mean values obtained with 0.005% and 0.01% BAC100, BAC58, BAC32, BOB, and Cet.

H2

O2

Production with DCFH-DA Test

Significant ROS production was observed with all the quater-nary ammoniums tested, even at lowest concentrations such as0.00001% (Fig. 2). Maximum production was observed at0.001% for BAC100 (mean fluorescence, 169% of the control),BAC58 (mean fluorescence, 220% of the control), BAC32 (meanfluorescence, 176% of the control), and Cet (mean fluores-cence, 235% of the control). At higher concentrations, H2O2

FIGURE 1. Membrane integrity evaluation with neutral red test after treatment with different concentrations of various quaternary ammo-niums. ( A ) Fifteen minutes of treatment: There was a significant de-crease of membrane integrity after 0.005% and 0.01% treatments. ( B )Fifteen minutes of treatment followed by 24 hours of cell recovery:Significant cellular damage was found at concentrations of 0.001% andhigher. ( A , B ) * P 0.001 compared with control.

644 Debbasch et al. IOVS, March 2001, Vol. 42, No. 3


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production decreased, possibly because of the cytotoxicity demonstrated by cell viability analysis. The maximum observed

with BOB appeared at 0.01% (mean fluorescence, 255% of thecontrol). Clb, EDTA, organomercurials, and MPHB also showedsignificant H2O2 production, but the maximal production wasobserved with thi (Fig. 3).

O2

. Production with Hydroethidine Test

Quaternary ammoniums induced an O2. synthesis at 0.005%

and 0.01% with no difference among the three BAC formula-tions, BOB, and Cet, according to ANOVA. The maximum wasobserved for all the quaternary ammoniums at 0.01%: 172%

with BAC100, 201% with BAC58, 172% with BAC32, 172% with BOB, and 159% with Cet (Fig. 4). Significant O2

. production was also observed with thi at 0.004% (mean fluorescence,124%, P 0.001 compared with control) and clb at 0.05%(mean fluorescence, 126%, P 0.001 compared with control),

but it was significantly less than the production observed with quaternary ammoniums ( P 0.0004 with thi compared with quaternary ammoniums; P 0.005 with clb compared with quaternary ammoniums; Fig. 5).

DNA Condensation Evaluation

All quaternary ammoniums and clb molecules induced a con-centration-dependent increase of the fluorescence ratio after 15 minutes of treatment, although all the quaternary ammoni-ums tested did not produce the same intensity of fluorescencecompared with control (Figs. 6A, 6B). Mean fluorescence in-creased from 122% to 547% with BAC100, from 129% to 452%

with BAC58, from 100% to 361% with BAC32, from 109% to367% with BOB, from 119% to 360% with Cet, and from 150%to 260% with clb. Chromatin condensation observed with allthe quaternary ammoniums tested at 0.005% and 0.01% wassignificantly higher than that with clb (mean fluorescence,370% with quaternary ammoniums, 162% with clb; P 0.0001

for all concentrations tested). As shown in Figure 7 with Hoechst staining, cells treated with quaternary ammoniumsshowed, in a concentration-dependent manner, chromatincondensation and fragmentation typical of apoptosis, whencompared with control cells. We present in Table 2 the mean

values obtained with all the concentrations tested for organo-mercurials, MPHB, and EDTA (no significant difference amongthe five concentrations tested and among the four preserva-tives, according to ANOVA). The chromatin condensation ob-served was significantly less than that observed with the qua-ternary ammoniums molecules (mean fluorescence, 119% with EDTA and MPHB and 128% with organomercurials versus 370%

with quaternary ammoniums, with P 0.0022 when EDTA or MPHB were compared with quaternary ammoniums and P 0.0002 when organomercurials were compared with quater-

nary ammoniums). After 24 hours of cell recovery (Fig. 6C), the quaternary

ammoniums still induced a marked increase of Hoechst fluo-rescence at 0.001% and higher, ranging from 165% with BAC100 to 415% with BAC32. No significant difference wasobserved between the five quaternary ammoniums. Quater-nary ammoniums at 0.005% and 0.01% induced a significantchromatin condensation compared with EDTA, organomercu-rials, and MPHB (mean fluorescence, 299% with quaternary ammoniums at all concentrations of 0.005% and 0.01%, 121%

with EDTA, 118% with organomercurials, 117% with MPHB,

FIGURE 2. H2O2 production evaluation after 15 minutes of treatment with quaternary ammoniums. * P 0.001 compared with control.

FIGURE 3. H2O2 production evaluation after 15 minutes of treatment with preservatives. * P 0.001 compared with control. Bar shading : 1,1/1000 dilution; 2, 1/100 dilution; 3, 1/10 dilution; 4, 1/2 dilution; and5, the higher concentration tested for all preservatives: 0.004% for thi,0.001% for PM, 0.03% for MPHB, 0.05% for Clb, and 0.01% for EDTA.

FIGURE 4. O2. production evaluation after 15 minutes of treatment

with quaternary ammoniums. * P 0.001 compared with control.

T ABLE 1. Membrane Integrity Evaluations

15-Minute Treatment

15-Minute Treatment

Followed by 24 Hours of

Cell Recovery

Control ( n 60) 100 100Quaternary ammoniums,

0.005 and 0.01% ( n 60) 24 4.3* 28 3.8*Chlorobutanol ( n 60) 112 4.3 97 10.7EDTA ( n 60) 107 9.5 92 16.3Organomercurials ( n 120) 110 16.3 91 18.8MPHB ( n 60) 113 7.5 101 11.3

Data are expressed as mean percentages of control ( SD). Qua-ternary ammoniums: means of BAC, BOB, and Cet; organomercurials:means of thi and PM; clb, EDTA, organomercurials, MPHB: means of allconcentrations tested.

* P 0.0001 compared with control and all other preservatives.No difference between all other preservative groups.



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P 0.0001 for all values). The chromatin condensation ob-served with clb was less significant (mean fluorescence, 241%, P 0.05) compared with quaternary ammonium molecules(Table 2).

Cell Size Analysis

Concentration-dependent toxicity was confirmed by flow cy-tometry by the alteration of cell size after quaternary ammo-nium treatment (Fig. 8). Cells treated with 0.01% BAC58 had a49% reduction of cell size in comparison with untreated cells.No difference was observed among the five quaternary ammo-niums. The other preservatives tested did not show any alter-ation of cell volume (data shown only for MPHB and threeconcentrations of BAC58 ).

DNA Content

We measured sub-G1 cell population after 15 minutes of treat-ment with the different preservatives. Normal untreated cellsshowed a 17% 5% sub-G1 population. The population of sub-G1 cells was 42% 3%, 54% 7%, and 74% 9% for

0.0001%, 0.001%, and 0.01% quaternary ammonium treat-ments, respectively (Fig. 9). The other preservatives tested didnot show any significant increase in the apoptotic cell popu-lation (sub-G1 population varied from 17% to 23%; data shownonly for MPHB and three concentrations of BAC58).

Morphologic Changes

A concentration-dependent cell retraction was observed after treatment with quaternary ammoniums molecules, whereas nomorphologic change was observed with the other preserva-tives tested (Fig. 10).

Correlation between the Different TestsPerformed on Preservatives

Membrane integrity and chromatin condensation showed sig-nificant negative correlation ( r 0.863, P 0.0001; Fig. 11).Poor but significant correlation was found between H2O2 pro-duction and membrane integrity or chromatin condensation(H2O2 production versus membrane integrity: r 0.417, P 0.0003; H2O2 production versus chromatin condensation: r 0.269, P 0.0104). However, this correlation was only dueto the two highest concentrations of quaternary ammoniumstested. A significant negative correlation was shown with mem-brane integrity and O2

. production ( r 0.551, P 0.0001).The results obtained with the neutral red probe after 15 min-utes of treatment were confirmed after 24 hours of cell recov-ery ( r 0.770, P 0.0001). At the same time, chromatincondensation was well correlated with the O2

. production ( r

FIGURE 5. O2. production evaluation after 15 minutes of treatment

with preservatives. * P 0.001 compared with control. Key to shadingis in Figure 3.

FIGURE 6. Chromatin condensation evaluation with Hoechst 33342test after treatment with different concentrations of various preserva-tives. Fifteen minutes of treatment with ( A ) quaternary ammoniums: asignificant concentration-dependent increase in fluorescence was ob-served, indicating an increase in chromatin condensation; ( B ) clb: asignificant increase in fluorescence was found for all concentrationstested; and ( C ) quaternary ammoniums followed by 24 hours of cellrecovery: chromatin condensation was still observed at 0.001% andhigher concentrations. * P 0.001 compared with control.



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0.738, P 0.0001) and was associated with the decrease of membrane integrity after 24 hours of cell recovery ( r 0.697, P 0.0001). H2O2 and O2

. productions, however, were not correlated.

Inhibition Study

No cytotoxicity was observed after a 1-hour vitamin E treat-ment (Fig. 12). Membrane integrity and chromatin condensa-tion were not altered, compared with complete culture me-dium alone. No ROS production was detected. After a 1-hour

vitamin E pretreatment followed by a 15-minute BAC 0.001%treatment, there was no alteration of membrane integrity (mean fluorescence, 88% with BAC 0.001% versus 120% with a

vitamin E pretreatment followed by a BAC treatment; P

0.001 compared with BAC). Significant decreases in chromatincondensation, H2O2, and O2

. production were observed com-pared with BAC. However, after a vitamin E pretreatmentfollowed by a BAC treatment, H2O2 production was increased(mean fluorescence, 146% with vitamin E with BAC versus105% with BAC alone; P 0.001 compared with BAC),

whereas no significant difference was observed when O2. pro-

ductions were compared.

DISCUSSION

Cationic agents are used in pharmaceutical preparations for antimicrobial preservation because of their ability to lyse mi-

crobial cellular membranes. The quaternary ammonium cat-ionic surfactants (BAC, BOB, Cet) we tested have detergent-likeproperties that may cause cell damage by emulsification of thecell wall lipids. Numerous clinical and biologic side effects of surfactant preservatives have been described, such as ocular irritation (with lacrimation, hyperemia, photophobia, andedema), punctate keratitis, gray corneal epithelial haze,pseudomembrane formation, decreased corneal epithelial mi-crovilli, and cytotoxicity to the corneal epithelial cells.1,2,5 Inaddition, cell permeability caused by quaternary ammoniums ispotentiated when EDTA is used. BAC disrupts the bacterialexternal membrane, and EDTA disorganizes the cell enve-lope.38

Organomercurials include thi, phenylmercuric nitrate or

acetate, and mercuric oxide. They bind to the cell membraneprotein sulfhydryl groups, causing an increase in cellular per-meability. Adverse ocular side effects due to these preserva-tives are rare. The most striking side effect is mercurial depos-its in various ocular tissues. These compounds have alsoinduced allergic reactions in sensitized persons; erythema,edema, and hyperemia of the eyelids; or conjunctivitis. Thi,however, has been shown to produce cytotoxicity for cornealepithelial cells.39–44

Clb is mainly known to induce cytotoxicity in corneal epi-thelial cells. In previous studies, it was reported that occasionaluse (twice daily up to 12 days) of a clb-preserved artificial tear resulted in only modest exfoliation of corneal epithelial cells(i.e., up to a maximum of 14%).45 These changes were revers-ible, and it was therefore suggested that the eye could adapt to

repeated use of these preserved artificial tears.46,47

Our results revealed new aspects of preservative toxicity. After a 15-minute application of all the quaternary ammoniumstested, membrane integrity of treated cells was altered,

whereas there was no variation of membrane integrity with theother preservatives. The difference was significant among allthe quaternary ammoniums tested at 0.005% and 0.01% andamong the other preservatives, even at the highest concentra-tions ( P 0.002 compared with quaternary ammoniums).

After a 0.01% quaternary ammonium treatment, cells showedcharacteristics of immediate abundant lysis, with membranedebris and low cell size on flow cytometric analysis graphs.The effects of all the quaternary ammoniums tested wereprogressive. There was a 75% decrease of membrane integrity

FIGURE 7. Hoechst 33342 nuclear staining of the cultured cells. ( A )Normal cell nuclei. Nuclei of cellstreated with ( B ) 0.03% MPHB: nomodifications compared with con-trol; ( C ) 0.00001%, ( D ) 0.005%, and( E ) 0.01% BAC showing a character-istic apoptotic peripheral condensa-tion and fragmentation of chromatin,in a concentration-dependent man-ner. Magnification, 40.

T ABLE 2. Chromatin Condensation Evaluations

15-Minute Treatment

15-Minute Treatment

Followed by 24 Hours of

Cell Recovery

Control ( n 60) 100 100Quaternary ammoniums,

0.005 and 0.01% ( n 60) 370 18.2* 299 19.3*Clb ( n 60) 162 16.3* 241 20.7*EDTA ( n 60) 119 10.2 121 10.4Organomercurials ( n 120) 128 18.1 118 17.6MPHB ( n 60) 119 18.0 117 17.7

See Table 1 for explanation of data.



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after a treatment with quaternary ammoniums at 0.005% and0.01% that persisted after 24 hours. No significant difference

was observed among the five quaternary ammoniums analyzed,and the three BAC tested were similar despite supposed differ-

ences in their antimicrobial activities. In contrast, the other preservatives tested showed no alteration of membrane integ-rity, even at high concentrations and even after 24 hours of cellrecovery.

FIGURE 8. Flow cytometric analysisof cell size after 15 minutes of treat-ment: ( A ) Control, ( B ) MPHB 0.03%,( C ) BAC 104%, ( D ) BAC 103%, and( E ) BAC 102%. FS, forward scatter.

FIGURE 9. Flow cytometric analysisof DNA content 15 minutes after celltreatments: ( A ) Control (untreatedcells) DNA. The number of cells isrepresented as a function of fluores-cence (FL). The percentage of apo-ptotic cells (sub-G1 population) de-tected was ( A ) 20%, control; ( B ) 23%,MPHB 0.03%; ( C ) 46%, BAC 104%;( D ) 53%, BAC 103%; and ( E ) 79%,BAC 102%.



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However, ROS evaluation showed that H2O2 productionincreased with the noncytotoxic preservatives and with qua-ternary ammonium concentrations less than or equal to0.001%, even though there was no alteration of cell viability,

whereas H2O2 production was decreased with quaternary am-

moniums at high concentrations (0.005% and 0.01%), possibly because of the decrease of cell viability observed for these twoconcentrations. Concerning O2

., there was a marked increasein production with all quaternary ammoniums tested at 0.005%and 0.01%, whereas a nonsignificant increase was observed atthe lowest concentrations. O2

., but not H2O2, could thereforeplay a role in the decrease of membrane integrity, becausethese two parameters were well correlated. Furthermore, O2

.

was also associated with chromatin condensation—these twoparameters being very significantly correlated—suggesting thatO2

. may induce apoptosis or at least participate in epithelialcell degeneration. In a cardiomyocyte model, ROS clearly in-duced apoptosis.48

Superoxide-generating systems have been demonstrated tobe cytotoxic for cultured cells, to degrade polysaccharides andDNA, to promote peroxidation of membrane lipids, to alter

FIGURE 10. F-actin exploration us-ing phalloidin. ( A ) Control cells andcells treated with ( B ) BAC 104%,( C ) BAC 102%, and ( D ) Cet 102%.

A concentration-dependent decreaseof cell size associated with chromatincondensation and cell disorganiza-tion was observed with quaternary ammoniums tested at 102%. Magni-fication, 1000.

FIGURE 11. Correlations between the different tests performed. Theresults obtained with all the preservatives tested are presented. Thedifferent test compared are RN (neutral red): membrane integrity evaluation; H2O2: hydrogen peroxide production evaluation; Hoechst:chromatin condensation evaluation; and O2

.: peroxide anion produc-tion evaluation. The decrease of RN is correlated with an increase of O2

. production and with an increase of Hoechst fluorescence. H2O2 variations cannot be interpreted.

FIGURE 12. Evaluation of membrane integrity, chromatin condensa-tion, and ROS production after a 1-hour vitamin E treatment and a1-hour vitamin E treatment followed by a 15-minute BAC 0.001%treatment. *** P 0.0001; ** P 0.001; * P 0.05 compared with BAC0.001%.



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